1. Field of the Invention
The present invention relates to compositions and methods useful in the preparation of major outer membrane protein ("MOMP") antigens of Haemophilus influenzae type b ("Hib"). In particular, the present invention is directed to DNA segments encoding Hib major outer membrane protein antigens, cells transformed with MOMP DNA segments, and the use of these in the preparation of MOMP antigen compositions.
2. Description of the Related Art
Haemophilus influenzae type b (Hib) is a leading cause of bacterial meningitis and other invasive infections in infants and young children. Children of the susceptible age range of 6 months to 4 years generally lack antibodies to the Hib capsular polysaccharide, which is a target for antibodies protective against systemic Hib disease. Moreover, children under the age of 2 years typically respond poorly to currently available polysaccharide-based vaccines such as pneumococcal vaccine or the Hib vaccine. For this reason, vaccines containing only capsular polysaccharides are of limited utility in the case of young children, the group most at risk for severe Hib infections.
Although children are generally poor responders to vaccines containing only capsular polysaccharides, it has been reported that children in this age group respond well to protein-based immunogens (1). For example, the outer membrane proteins of Hib appear to be immunogenic in infants, as exemplified by reports of antibody development to Hib cell surface exposed outer membrane proteins following systemic Haemophilus disease (2). Moreover, researchers have reported that passive administration of antibodies directed against non-capsular Hib antigens served to protect against experimental Hib bacteremia. (3).
A number of Hib protein components have been studied as possible candidates for the production of passive immunoreagents or in the development of vaccines. While some Hib proteins are either insufficiently antigenic, or their corresponding antibody non-protective (4), one class of Hib proteins have shown some initial promise. These proteins, the so-called major outer membrane proteins or "MOMPs" are proteins that are localized to the Hib outer membrane fractions and thus, are more likely to be exposed or available for antibody binding than are more internally localized proteins. In particular, experiments have been reported wherein antibodies directed against Hib outer membrane proteins appeared to confer protection against bacteremia following intraperitoneal challenge with Hib, whereas antibodies against lipopolysaccharide components lacked protective activity (3). In these experiments, antibodies directed against several outer membrane proteins were detected in the protective immune sera by whole cell radio-immunoprecipitation, suggesting the availability of these antigens on the surface of encapsulated organisms.
In general, the surface-exposed outer membrane proteins of Haemophilus influenzae type b studied to date which may have potential for vaccine development, can be characterized as follows--the 98K protein; the P1 protein which is heat modifiable and exhibits an apparent molecular weight of 45-50K after heating at 100.degree. C. in 2% sodium dodecyl sulfate (this protein is also called protein a); the P2 protein which is a porin and which has an apparent molecular weight of 38K-40K (this protein is also called protein b/c or the 39K/38K protein); and the P6 protein which exhibits an apparent molecular weight of 14K-16K.
One of the more interesting Hib MOMP antigens, from a vaccine component or passive immunotherapy standpoint, is the antigen group referred to variously as the 39K (5) or P2 (6) protein species. This protein has been identified as a porin and forms non-covalent complexes with Hib lipopolysaccharide (7,8a). The P2 protein, in fact, demonstrates size heterogeneity from strain to strain, ranging from approximately 38K to 40K, depending on the strain and the method used for determining molecular weight.
The P2 antigen is of particular interest in that studies have demonstrated that polyclonal serum antibodies directed against this antigen are capable of protecting infant rats against Hib bacteremia (6). Unfortunately, while this antigen(s) appears to be useful and desirable from a medical and commercial standpoint, its availability from currently available natural sources is limited. For example, although the protein is a membrane component of Hib strains, its isolation from these strains generally results in limited recoveries.
Thus, there is currently a need for alternative sources from which individual Hib protein antigens and, in particular, selected MOMP antigens such as the P2 antigens, may be obtained and purified. Ideally, such sources should not only allow the preparation of improved anti-Hib antibody and immunogen compositions in terms of ease of preparation and purity, but should also allow isolation of larger, more commercially reasonable quantities.